Peptides, in particular oligopeptides have many applications, for instance as pharmaceutical, food or feed ingredient, or cosmetic ingredient.
Processes for synthesizing (oligo)peptides are generally known in the art. Oligopeptides can be chemically synthesized in a stepwise fashion in solution or on the solid phase via highly optimized processes. However, peptides longer than 10-15 amino acids are often very difficult to synthesize due to side reactions and as a consequence purification is troublesome. Therefore, peptides longer than 10 amino acids are often synthesized by a combination of solid-phase synthesis of side-chain protected oligopeptide fragments which are subsequently chemically condensed in solution, e.g. as in a 10+10 condensation to make a peptide of 20 amino acids. The major drawback of chemical side-chain protected oligopeptide fragment condensation is that upon activation of the C-terminal amino acid residue of the acyl donor racemisation occurs. In contrast, enzyme-catalysed peptide couplings are completely devoid of racemisation and have several other advantages over chemical peptide synthesis such as the absence of side reactions on the side-chain functionalities. For industrial application, an enzymatic peptide synthesis concept based on a kinetic approach, i.e. using an acyl donor C-terminal ester is most attractive (see for instance N. Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology”, 1st reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).
Chemo-enzymatic peptide synthesis can entail the enzymatic coupling of oligopeptide fragments which have individually been synthesized using chemical synthesis, fermentation, or by a combination of chemical and enzymatic coupling steps. Some reports have been published on the enzymatic condensation of oligopeptide fragments in aqueous solution (Kumaran et al. Protein Science, 2000, 9, 734; Bjorup et al. Bioorg. Med. Chem. 1998, 6, 891; Homandberg et al. Biochemistry, 1981, 21, 3387; Komoriya et al. Int. J. Pep. Prot. Res. 1980, 16, 433).
It was found by Wells et al. (U.S. Pat. No. 5,403,737) that the condensation of oligopeptides in aqueous solution could be significantly improved by altering the active site of subtilisin BPN′, a subtilisin from B. amyloliquefaciens (SEQUENCE ID NO: 2). When two mutations were introduced, i.e. S221C and P225A, a subtilisin BPN′ variant called subtiligase was obtained having a 500-fold increased synthesis over hydrolysis ratio (S/H ratio) as compared to wild-type subtilisin BPN′. In further experiments Wells et al. added five additional mutations to subtiligase, i.e. M50F, N76D, N109S, K213R and N218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). The new mutant called stabiligase appeared moderately more resistant to sodium dodecasulphate and guanidinium hydrochloride, but hydrolysis was still a major side reaction. For instance an (oligo)peptide carboxyamidomethyl-ester (Cam-ester) was ligated to an (oligo)peptide amine using stabiligase in a yield of 44%. In this example, 10 equivalents of the (oligo)peptide C-terminal ester were used and thus, 9.56 equivalents of the (oligo)peptide C-terminal ester were hydrolyzed at the C-terminal ester functionality and only 0.44 equivalents ligated to the (oligo)peptide amine to form the product. Probably for this reason, the past 20 years subtiligase nor stabiligase have been industrially applied in enzymatic peptide synthesis, to the best of the inventors knowledge.
In post-published WO 2016/056913 (claiming priority of PCT/NL2014/050707) a solution is provided for the undesirably high hydrolytic activity encountered with enzymes like subtiligase or stabiligase when used for (oligo)peptide synthesis in an aqueous environment, by providing a subtilisin BPN′ variant or a homologue thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homologue sequence thereof:                a deletion of the amino acids corresponding to positions 75-83;        a mutation at the amino acid position corresponding to S221, the mutation being S221C;        a mutation at the amino acid position corresponding to P225, said mutation being P225A;        
The present inventors realized that for enzymatic (oligo)peptide synthesis to obtain a certain peptide product at will there is not only room for improvement by identifying enzymes which have a good synthesis over hydrolysis ratio, but also in selecting which (oligo)peptide fragments to use for assembling the (oligo)peptide of interest. As will be understood by the skilled person, from the amino acid sequence of the (oligo)peptide it can be determined which different fragments (two or more) can at least theoretically be coupled together in the right order to result in the (oligo)peptide of interest. However, from the amino acid sequence of the (oligo)peptide for the enzymatic coupling(s) as such it does generally not follow which coupling position or positions would be optimal, especially not if the (oligo)peptide is large, e.g. having 8 or more, in particular 12 or more, more in particular 20 or more amino acid units. Designing a desirable coupling strategy, which should offer a synthesis process with satisfactory selectivity and coupling yield requires determining the number of fragments and the length of each of the fragments (which define the coupling position) to be used in the enzymatic synthesis, which development is therefore a complicated, often lengthy, task requiring multiple trial-and-error approaches.
Specific examples of oligopeptides for which it would be desired to design an enzymatic synthesis process include Exenatide, Thymosin alpha 1 and Lixisenatide. Exenatide is an oligopeptide that can be used as adjunctive therapy to improve glycemic control in patients with type 2 diabetes mellitus who are taking metformin, but have not achieved adequate glycemic control.
Exenatide is difficult to prepare via classical chemical synthesis since it is a long oligopeptide, i.e. having 39 amino acids, and is virtually impossible to produce via known fragment condensation methodology due to racemisation, since there are no Gly or Pro residues present at strategic positions. Generally, the full solid-phase-synthesis of a 39 amino acid long peptide results in purified yields of around 10-20%, corresponding to 95-96% yield per step. Due to the inefficient synthesis of Exenatide (10-15% overall yield on large scale) cost prices for this medicine are extremely high.
Similar problems with known synthesis methodology apply to the synthesis of Lixisenatide, a variant of Exenatide with increased water-solubility, having 44 amino acids. The overall yield for Lixisenatide is even worse and cost-prices are even higher.
Thymosin alpha 1 is an enhancer of cell-mediated immunity. It does not contain any Gly or Pro residue's and is thus impossible to produce chemically (non-enzymatically) via fragment condensation. Thymosin alpha 1 is a classic example of a peptide that is extremely difficult to produce via full solid phase peptide synthesis due to hydrophobic collapse. When standard solid phase methods are applied for the synthesis of the 28 amino acid long Thymosin alpha 1, crude yields of 10% have been reported (Fernando Albericio, Journal of Peptide Science, 2009, 92, 565-572). On large scale, three consecutive preparative HPLC purifications are needed to obtain an acceptable purity of the product.
Clearly, there is a need for new technologies to improve the synthesis, overall yield and cost-prices of many pharmaceutical peptides, such as Exenatide, Lixisenatide, Thymosin alpha 1 and analogues thereof.
The inventors further realized that there is a need for an improved method of designing a process for the enzymatic synthesis of a cyclic (oligo)peptide, since from the amino acid sequence of a cyclic (oligo)peptide it is generally not evident which non-cyclic (oligo)peptide would be enzymatically cyclized adequately by coupling its C-terminal end and N-terminal end to form a peptide bond; after all the number of non-cyclic (oligo)peptides that have an amino acid sequence from which the cyclic (oligo)peptide can (conceptually) be composed by cyclisation of both ends is typically equal to the number of peptide bonds in the cyclic (oligo)peptide.